Using Drosophila larval imaginal discs to study low-dose radiation-induced cell cycle arrest.
Using Drosophila larval imaginal discs to study low-dose radiation-induced cell cycle arrest.
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DOI：
10.1007/978-1-61779-273-1_8
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Li, Willis X
中科院分区：
文献类型：
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作者：
Yan, Shian-Jang;Li, Willis X
在遗传毒性应激下，细胞周期检查点反应的激活导致细胞周期停滞，这允许细胞在继续循环之前修复DNA损伤。果蝇幼虫上皮囊，称为成虫盘，是研究辐射诱导细胞周期阻滞的一个很好的体内模型系统。幼虫成虫盘在受到低剂量辐射后进入细胞周期停滞，易于遗传操作，对于生物体的存活不是至关重要的，并且可以容易地解剖用于进一步的分子或细胞分析。在本章中，我们描述了评估低剂量辐射诱导的细胞周期阻滞的方法。通过有丝分裂标记磷酸化组蛋白H3（磷酸化组蛋白H3或pH 3）的免疫荧光染色鉴定有丝分裂细胞。当无转基因表达的游走的三龄对照幼虫暴露于500拉德的X射线或γ射线照射时，翅成虫盘中的pH 3阳性细胞的数量从照射前的数百个减少到照射后的约30个，在前室和后室之间具有相等的分布（Yan等人，2011，FASEB J）。使用GAL 4/UAS系统，RNAi、cDNA或microRNA海绵转基因可以使用驱动器（如enrailed（en）-Gal 4）在翼盘的后室中表达，而前室用作内部对照。这种方法使得有可能做全基因组的遗传筛选参与辐射诱导的细胞周期停滞的分子。
Under genotoxic stress, activation of cell cycle checkpoint responses leads to cell cycle arrest, which allows cells to repair DNA damage before continuing to cycle. Drosophila larval epithelial sacs, called imaginal discs, are an excellent in vivo model system for studying radiation-induced cell cycle arrest. Larval imaginal discs go into cell cycle arrest after being subjected to low-dose irradiation, are subject to easy genetic manipulation, are not crucial for survival of the organism, and can be dissected easily for further molecular or cellular analysis. In this chapter, we describe methods for assessing low-dose irradiation-induced cell cycle arrest. Mitotic cells are identified by immunofluorescence staining for the mitotic marker phosphorylated histone H3 (phospho-histone H3 or pH3). When wandering third-instar control larvae, without transgene expression, are exposed to 500 rads of X-ray or γ-ray irradiation, the number of pH3-positive cells in wing imaginal discs is reduced from hundreds before irradiation to approximately 30 after irradiation, with an equal distribution between the anterior and posterior compartments (Yan et al., 2011, FASEB J). Using the GAL4/UAS system, RNAi, cDNA, or microRNA sponge transgenes can be expressed in the posterior compartment of the wing disc using drivers such as engrailed (en)-Gal4, while the anterior compartment serves as an internal control. This approach makes it possible to do genome-wide genetic screening for molecules involved in radiation-induced cell cycle arrest.